ADP-ribosyltransferase (ADPRT) catalyzes the reaction in which the ADP-ribose moiety of ${eta}-NAD^+$ is transferred to specific amino acid residues in target proteins. The ADPRT of Mycobacterium smegmatis has been known to inactivate rifampin through ADP-ribosylation. However, the enzymatic characteristics and functions of the enzyme have not been elucidated yet. In this study, the ADPRT-glutathione S-transferase (GST) fusion protein was expressed in Escherichia coli and enzymatic characteristics of the fusion protein were investigated. ADPRT-GST fusion protein was an ADP-ribosyltransferase that had no NAD glycohydrolase activity. ADPRT-GST fusion protein showed no self-inactivation phenomenon that is a universal nature for all NAD glycohydrolases and is important in regulating its activity. ADPRT activity of the enzyme was decreased by novobiocin and isonicotinic acid hydrazide. These results suggest that Mycobacterium smegmatis ADPRT could be regulated by a different way from other NADases and involved in bacterial physiological process through a post-translational modification of cytosolic proteins.